Screening for hepatitis c virus entry inhibitors

ABSTRACT

The present invention features methods of screening for compounds that inhibit HCV binding to a cell, methods of inhibiting IICV entry into a cell, and methods of actively or prophylactically treating against an IICV infection. The different methods are based on the identification of the scavenger receptor class B type I as a target site for HCV E2 binding to a cell.

RELATED APPLICATIONS

The present application claims priority to provisional application U.S.Ser. No. 60/344,504, filed Nov. 9, 2001, which is hereby incorporated byreference herein.

BACKGROUND OF THE INVENTION

The references cited in the present application are not admitted to beprior art to the claimed invention.

It is estimated that about 3% of the world's population is infected withthe hepatitis C virus (HCV). (Wasley et al., Semin. Liver Dis. 20:1-16,2000.) HCV exposure results in an overt acute disease in a smallpercentage of cases, while in most instances the virus establishes achronic infection causing liver inflammation and slowly progresses intoliver failure and cirrhosis. (Strader et al., ILAR J. 42:107-116, 2001.)Epidemiological surveys indicate an important role for HCV in the onsetof hepatocellular carcinoma. (Strader et al., ILAR J. 42:107-116, 2001.)HCV can be classified into a number of distinct genotypes (1 to 6), andsubtypes (a to c). The distribution of the genotypes and subtypes variesboth geographically and between risk groups. (Robertson et al., ArchVirol. 143:2493-2503,1998.)

The HCV genome consists of a single strand RNA about 9.5 kb encoding aprecursor polyprotein of about 3000 amino acids. (Choo et al., Science244:362-364, 1989, Choo et al., Science 244:359-362, 1989.) The HCVpolyprotein contains the viral proteins in the order:C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NSSB. Cleavage of the precursorpolyprotein results in mature structural and non-structural viralproteins. (Neddermann et al., Biol. Chem. 378:469476, 1997.)

As part of its infection cycle, HCV enters into a cell. The LDL receptorand CD81 molecule have been identified as putative HCV receptors. TheLDL receptor has been suggested to mediate virus internalization viabinding to LDL particles that are virus-associated. (Agnello et al.,Proc. Natl. Acad. Sci. U.S.A. 96:12766-12771, 1999.) The CD81 moleculehas been suggested to bind HCV E2 based on recombinant envelope proteinE2 from HCV genotype 1a. (Pileri et al., Science 282:938-941, 1998.)

SUMMARY OF THE INVENTION

The present invention features methods of screening for compounds thatinhibit HCV binding to a cell, methods of inhibiting HCV entry into acell, and methods of actively or prophylactically treating against anHCV infection. The different methods are based on the identification ofthe scavenger receptor class B type I (SR-BD as a target site for HCV E2binding to a cell.

Targeting the SR-BI to inhibit HCV entry into a cell can be achieved byinhibiting one or more of the following: (a) activities relating to HCVbinding to SR-BI, (b) activities related to HCV internalization mediatedby SR-BI, including activities downstream from SR-BI binding to HCV, and(c) activities related to functional surface expression of SR-BI.

Thus, a first aspect of the present invention features a method ofscreening for a compound that inhibits the ability of a HCV E2polypeptide to bind to a cell. The method involves contacting SR-BI or afunctional derivative thereof with a polypeptide that binds to the SR-BIHCV E2 binding site and with a test compound, and measuring the abilityof the test compound to inhibit binding of the polypeptide to SR-BI orthe functional derivative thereof.

A “compound” or “test compound” refers to a discrete chemical entity.The term compounds includes molecules of different sizes andcompositions. Examples of compounds include small molecules, peptides,polypeptides, antibodies, and nucleic acid.

The “SR-BI HCV E2 binding site” is the site to which at least the HCV E2polypeptide from HCV 1a binds SR-BI. An example of such an HCV E2polypeptide from HCV 1a is provided in the Examples infra. Reference tothe ability of HCV 1a to bind SR-BI does not exclude binding of HCV E2from other HCV strains to SR-BI. For example, HCV E2 from other HCVstrains such as HCV 1b can bind to the naturally occurring human SR-BIHCV E2 binding site.

SR-BI and functional derivatives of SR-BI contain a SR-BI amino acidsequence region of at least 20 contiguous amino acids as that present inSEQ. ID. NO. 1 and can bind at least HCV E2 from HCV 1a.

SEQ. ID. NO. 1 provides a human SR-BI sequence. The presence of at least20 continuous amino acids as provided in SEQ. ID. NO. 1 provides astructural tag distinguishing SR-BI or a functional derivative thereoffrom other proteins.

Reference to “inhibit” or “inhibiting” indicates a detectable reductionin activity. Preferably, there is at least about a 50%, at least about75%, or at least about 95% percent reduction in activity.

Another aspect of the present invention describes a method of screeningfor a compound that inhibits SR-BI activity. The method involves thesteps of. (a) contacting a cell capable of expressing a SR-BI or afunctional derivative thereof with a polypeptide that binds to the SR-BIHCV E2 binding site and with a test compound, and (b) measuring theability of the test compound to inhibit one or more of the following:(i) activities related to HCV binding to SR-BI or a functionalderivative thereof, (ii) activities related to HCV internalization, and(iii) functional surface expression of SR-BI or a functional derivativethereof.

In an embodiment of the present invention, the test compound ispre-incubated with SR-BI prior to adding the polypeptide that binds tothe SR-BI HCV E2 binding site. Pre-incubation with a test compound is apreferred method for assaying SR-BI functional surface expressioninhibitors.

Reference to “capable of expressing” a polypeptide indicates that in theabsence of an expression inhibitor, the polypeptide will be expressed indetectable amounts and has detectable activity related to HCV binding orHCV internalization. Expression inhibitors include compounds such asantisense nucleic acid and ribozymes able to decrease activity ofnucleic acid encoding for SR-BI and compounds that can modulatefunctional surface expression of SR-BI at the transcriptional orpost-transcriptional levels.

Compounds modulating functional surface expression at thepost-transcriptional level include compounds acting on lipid raftsmembrane compartments (referred to as “raft domains”) to alter SR-BIactivity. SR-BI activity that can be altered by such compounds includeHCV binding and internalization.

Another aspect of the present invention features a method of inhibitingentry of a HCV into a cell. The method involves the step of contactingthe cell with a SR-BI E2 binding antagonist.

A “SR-BI E2 binding antagonist” can at least inhibit binding of anaturally occurring HCV E2 to the SR-BI of SEQ. ID. NO. 1. Preferably,the SR-BI E2 binding antagonist inhibits at least binding of HCV E2 fromHCV 1a.

Another aspect of the present invention features a method of treating anHCV infected patient. The method involves the step of decreasing SR-BIactivity or functional surface expression.

Other features and advantages of the present invention are apparent fromthe additional descriptions provided herein including the differentexamples. The provided examples illustrate different components andmethodology useful in practicing the present invention. The examples donot limit the claimed invention. Based on the present disclosure theskilled artisan can identify and employ other components and methodologyuseful for practicing the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate immunoblot detection of HCV E2 obtained fromgenotype 1a (FIG. 1A) and biotinylated cell surface proteins interactingwith HCV E2 (FIG. 1B). Biotinylated HepG2 cells were incubated inpresence (lanes 1 and 3) or absence of HCV E2 recombinant protein (lanes2 and 4). The bound species were cross-linked with DTSSP and thecomplexes were immunoprecipitated with an antibody against the His tagof the HCV E2 recombinant protein. Samples were eluted both undernon-reducing condition, (lanes 1 and 2) and reducing conditions (lanes 3and 4), that allowed the cleavage between the cross-linked molecularspecies, and loaded on 10% SDS-PAGE. In FIG. 1A, HCV E2 protein isdetected with anti-E2 rat mAb followed by anti-rat HRP conjugated as amonomer under reducing conditions (lane 3) and at higher molecularweight under non-reducing conditions (lane1). In FIG. 1B, the reactivitywith streptavidin HRP conjugated reveals under reducing conditions (lane3) a biotinylated protein band at 82 kDa.

FIG. 2 illustrates silver staining of a 7.5% SDS-PAGE loaded withsamples obtained after a purification step with Con-A sepharose and thedeglycosylation step with the PNG-ase F enzyme. The arrows show thepurified receptor migrating at 82 kDa (lane 1) before PNG-ase treatment(−) and migrating at 54 kDa (lane 2) after the deglycosylation (+). Inthe control samples cross-linking was performed in absence of HCV E2(lanes 3 and 4). Fetuin was loaded on the SDS-PAGE beforedeglycosylation and after (lane 5 and lane 6) as a control for thePNG-ase F enzymatic activity.

FIG. 3 illustrates a Western blot analysis of the HCV E2 receptorpurified on Con-A sepharose and deglycosylated with PNG-ase F (+).Rabbit anti-SR-BI polyclonal antibodies incubation was followed byanti-rabbit HRP conjugated for detection of both the glycosylatedCane 1) and deglycosylated (lane 2) receptor protein. Lanes 3 and 4represent the control experiment where cross-linking was performed inabsence of HCV E2.

FIGS. 4A and 4B illustrates a FACS analysis of the binding of HCV E2recombinant protein from strain H to mock transfected (FIG. 4A) or SR-BItransfected BHK-21 cells (FIG. 4B). Dot plot analysis showed that 10% ofthe cells transfected with pcDNA3-SR-BI show binding for HCV E2.

FIGS. 5A-F illustrate CHO cells transfected with the plasmid pcDNA3 andpcDNA3 encoding the human SR-BI or the mouse SR-BI. FIGS. 5A-Cillustrate FACS analysis of the anti-SR-BI (Novus Biologicals NB400-104) binding to CHO transfected cells. FIGS. 5D-F illustrate theanalysis of the E2 protein binding to transfected cells.

FIG. 6 illustrates competition of E2 binding to HepG2 cells and CHOcells transfected with human SR-BI (CHO-SR-BI) by the anti-HVR1 mAb9/27. Binding was detected by FACS analysis and expressed as percentageof the median fluorescence intensity values obtained in absence ofcompetitor. Binding to HepG2 (open triangle) and CHO-SR-BI (open square)of E2 from H isolate. Binding of E2 from BK isolate to HepG2 (filledtriangle) and to CHO-SR-BI (filled square). E2 recombinant proteins wereused at half saturating concentration.

DETAILED DESCRIPTION OF THE INVENTION

SR-BI is identified herein as a HCV receptor. SR-BI binding to HCV isindependent from CD81. Without being limited to any particular theory,SR-BI may provide a privileged HCV entry site by mediating E2 viralglycoprotein interaction with raft domains.

Naturally occurring SR-BI is highly expressed in the liver hepatocytesand steroidogenic tissues, and mediates the selective cellular uptake ofcholesterol and phospholipids. (Acton et al., Science 271:518-520, 1996,Urban et al., J. Biol. Chem. 275:33409-33415, 2000.) SR-BI and otherscavenger receptors recognize modified lipid particles both acetylatedLDL and oxidized LDL. In contrast to other scavenger receptors, SR-BIalso binds with high affinity to native HDL and LDL. (Acton et al.,Science 271:518-520, 1996.) SR-BI has been located into raft domains.Rafts domains are thought to represent a specific physical state oflipid bilayer, the liquid order phase. (Brown et al., Annu. Rev. Cell.Dev. Biol. 14:11-136, 1998, van der Goot et al., Semin. Immunol.13:89-97, 2001.) Proteins localized into raft domains are resistant tocold non-ionic detergent extraction (detergent resistant membranes).(London et al., Biochim. Biophys Acta. 1508:182-195, 2000.)

Proteins localized in raft domains are either GPI membrane anchored orfatty acylated. (Brown et al., Annu. Rev. Cell. Dev Biol. 14:11-136,1998.) SR-BI is a fatty acylated protein. (Babitt et al., J. Biol. Chem.272:13242-13249, 1997.)

Rafts domains may represent a preferential entry site for pathogensproviding them a way to escape from the classical degradative pathway.(van der Goot et al., Semin. Immunol. 13:89-97, 2001). Examples ofpathogens that may enter a cell by targeting raft domain componentsinclude SV40, echoviruses, HIV, and HTLV-1. (Bergelson et al., Proc.Natl. Acad. Sci. U.S.A. 91:6245-6249, 1994, Manes et al., EMBO Rep.1:190-196, 2000, Niyogi et al., J. Virol. 75:7351-7361, 2001, Parton etal., Immunol. Rev. 168:23-31, 1999, van der Goot et al., Semin. Immunol.13:89-97, 2001.)

The identification of SR-BI as a site for HCV E2 binding provides atarget that can be modulated to study the HCV infection cycle and toinhibit HCV replication or infection. The ability of a test compound tomodulate the interaction between SR-BI and HCV E2 can be performed forexample, using assays employing a naturally occurring SR-BI orderivative thereof that binds HCV E2, a compound that binds to the SR-BIHCV E2 binding site, and the test compound.

Test compounds found to inhibit HCV E2 interaction with SR-BI can beused, for example, to study the effect of modulating SR-BI HCV E2interaction on HCV replication or infection. Those test compounds havingappropriate pharmacological properties such as efficacy and lack ofunacceptable toxicity may be used to treat or inhibit HCV infection in apatient.

Scavenger Receptor Class B Type I (SR-BI)

SR-BI and functional derivatives thereof used to screen for modulatorsof SR-BI interaction with HCV E2 can bind at least HCV E2 from HCV 1 a.SR-BI and functional derivative of SR-BI contain a SR-BI amino acidsequence region of at least 20 contiguous amino acids as that present inSEQ. ID. NO. 1. The presence of at least 20 contiguous amino acids asprovided in SEQ. ID. NO. 1 provides a tag distinguishing SR-BI fromother proteins.

SR-BI can be obtained from mammalian sources such as human, hamster,mouse and rat. The ability of SR-BI obtained from a particular source tobind HCV E2 can be confirmed using techniques such as those described inthe Examples infra. Examples of naturally occurring SR-BI amino acid andnucleic acid sequences are provided for by SEQ. ID. NO. 1, SEQ. ID. NO.2, and in references such as Acton U.S. Pat. No. 5,998,141, Calvo etal., J. Biol. Chem. 268:18929-18935, 1993, Acton et al., J. Biol. Chem.269:21003-21009, 1994, Cao et al., J. Biol. Chem. 272:33068-33076, 1997,and Webb et al., J. Biol. Chem. 24:15241-15248, 1998.

Methods screening for compounds inhibiting the ability of a HCV E2polypeptide to bind to a cell, or inhibiting SR-BI activity, preferablyemploy human SR-B 1 or a functional derivative thereof. Human SR-BI isalso referred to in the literature as CLA-1. Splice variants orisoforms, and different polymorphic forms of SR-BI that bind to HCV E2are included within the definition of SR-BI by reference to the presenceof an at least 20 amino acid tag.

Based on SR-BI sequences known in the art, additional naturallyoccurring SR-BI encoding nucleic acid, preferably of human origin, canbe obtained. Cloning techniques well known in the art, such as thoseemploying probes, primers, and degenerative probes and primers, can beused to clone SR-BI.

Sets of degenerative probes and primers can be produced taking intoaccount the degeneracy of the genetic code. Hybridization conditions canbe selected to control probe or primer specificity to allow forhybridization to nucleic acids having similar sequences.

Techniques employed for hybridization detection and PCR cloning are wellknown in the art. Nucleic acid detection techniques are described, forexample, in Sambrook, et al., Molecular Cloning, A Laboratory Manual,2nd Edition, Cold Spring Harbor Laboratory Press, 1989. PCR cloningtechniques are described, for example, in White, Methods in MolecularCloning, volume 67, Humana Press, 1997.

A naturally occurring SR-BI that binds HCV E2 can be used to producefunctional variants. Variants include naturally occurring SR-BI with oneor more amino acid alterations. Amino acid alterations aresubstitutions, additions and deletions. SR-BI activity, such as bindingto HCV, SR-BI functional expression, and HCV internalization can bemeasured based on the guidance described herein.

Differences in naturally occurring amino acids R groups may be takeninto account in designing variants. An R group affects differentproperties of an amino acid such as physical size, charge, andhydrophobicity. Amino acids can be divided into different groups asfollows: neutral and hydrophobic (alanine, valine, leucine, isoleucine,proline, tyrptophan, phenylalanine, and methionine); neutral and polar(glycine, serine, threonine, tryosine, cysteine, asparagine, andglutamine); basic (lysine, arginine, and histidine); and acidic(aspartic acid and glutamic acid).

Generally, in substituting different amino acids it is preferable toexchange amino acids having similar properties. Substituting differentamino acids within a particular group, such as substituting valine forleucine, arginine for lysine, and asparagine for glutamine are goodcandidates for not causing a change in polypeptide functioning.

Changes outside of different amino acid groups can also be made.Preferably, such changes are made talking into account the position ofthe amino acid to be substituted in the polypeptide. For example,arginine can substitute more freely for nonpolar amino acids in theinterior of a polypeptide than glutamate because of its long aliphaticside chain. (See, Ausubel, Current Protocols in Molecular Biology, JohnWiley, 1987-1998, Supplement 33 Appendix IC.) SEQ. ID. NO. 1 provides areference sequence for SR-BI including SR-BI functional variants. Indifferent embodiments SR-BI or a functional variant thereof contains atleast contiguous 50 amino acids as that present in SEQ. ID. NO. 1,contains at least contiguous 75 amino acids as that present in SEQ. ID.NO. 1; contains SEQ. ID. NO. 1 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acid alterations; or contains a substantially similar sequence toSEQ. ID. NO. 1.

A “substantially similar sequence” indicates an identity of at leastabout 65% to a reference sequence. Thus, for example, polypeptideshaving an amino acid sequence substantially similar to SEQ. ID. NO. 1have an overall amino acid identity of at least about 65% to SEQ. ID.NO. 1. In different embodiments, substantially similar sequence refersto a sequence identity of at least about 75%, at least about 85%, atleast about 95%, or 100%.

Amino acid sequence identity can be determined by methods well known inthe art that compare the amino acid sequence of one polypeptide to theamino acid sequence of a second polypeptide and generate a sequencealignment. Amino acid identity can be calculated from the alignment bycounting the number of aligned residue pairs that have identical aminoacids.

Methods for determining sequence identity include those described bySchuler, G. D. in Bioinformatics: A Practical Guide to the Analysis ofGenes and Proteins, Baxevanis, A. D. and Ouelette, B. F. F., eds., JohnWiley & Sons, Inc, 2001; Yona et al., in Bioinformatics: Sequence,Structure and Databanks, Higgins, D. and Taylor, W. eds., OxfordUniversity Press, 2000; and Bioinformatics: Sequence and GenomeAnalysis, Mount, D. W., ed., Cold Spring Harbor Laboratory Press, 2001).Methods to determine amino acid sequence identity are codified inpublicly available computer programs such as GAP (Wisconsin PackageVersion 10.2, Genetics Computer Group (GCG), Madison, Wis.), BLAST(Altschul et al., J. Mol. Biol. 215(3):403-10, 1990), and FASTA(Pearson, Methods in Enzymology 183:63-98, 1990, R. F. Doolittle, ed.).

In an embodiment of the present invention sequence identity between twopolypeptides is determined using the GAP program (Wisconsin PackageVersion 10.2, Genetics Computer Group (GCG), Madison, Wis.). GAP usesthe alignment method of Needleman and Wunsch. (Needleman et al., J. Mol.Biol. 48:443-453, 1970.) GAP considers all possible alignments and gappositions between two sequences and creates a global alignment thatmaximizes the number of matched residues and minimizes the number andsize of gaps. A scoring matrix is used to assign values for symbolmatches. In addition, a gap creation penalty and a gap extension penaltyare required to limit the insertion of gaps into the alignment. Defaultprogram parameters for polypeptide comparisons using GAP are theBLOSUM62 (Henikoff et al., Proc. Natl. Acad. Sci. USA, 89:10915-10919,1992) amino acid scoring matrix (MATrix=blosum62.cmp), a gap creationparameter (GAPweight=8), and a gap extension pararameter(LENgthweight=2).

Polypeptide Bindings to the SR-BI HCV E2 Binding Site

Polypeptides capable of binding to the SR-BI HCV E2 binding site containa region able bind to the same site as HCV E2. The polypeptide regionthat binds to SR-BI includes naturally occurring E2 regions or bindingfragments thereof, derivatives of such E2 regions or binding fragmentsthereof, and E2 mimotopes.

Polypeptides capable of binding to the SR-BI HCV E2 binding site canalso contain regions not involved in SR-BI binding. Non-binding regions,if present, do not prevent the polypeptide from binding to at least thehuman SR-BI of SEQ. ID. NO. 1. Preferred non-binding regions areadditional HCV regions and regions that facilitate detection of binding.

Regions facilitating detection of binding include detectable labels andregions that can be detected. Examples of detectable labels includemoieties such as radiolabels, luminescent molecules, haptens and enzymesubstrates. Examples of regions that can be detected include regionsthat provide an epitope for antibody binding or that provide a specificbinding region for other types of compounds.

HCV E2 binding mimotopes have a primary structure unrelated to HCV E2,but share binding characteristics with HCV E2. References describingtechniques for producing mimotopes in general and describing differentHCV E2 mimotopes include Felici et al., U.S. Pat. No. 5,994,083 andNicosia et al., International Application Number WO 99/60132.

The ability of a polypeptide to bind to the SR-BI E2 binding site can bedetermined, for example, by competition experiments with a HCV E2polypeptide already shown to bind to SR-BI. Such experiments can beperformed employing a polypeptide that may bind to the SR-BI HCV E2binding site as a test compound.

Recombinant SR-BI Expression

Screening for compounds inhibiting HCV binding to SR-BI is facilitatedusing recombinant nucleic acid expressing SR-BI or a functionalderivative thereof. Recombinantly expressed receptors offers severaladvantages in screening for compounds active at a polypeptide, such asthe ability to express the polypeptide in a cell having little or noendogenous expression of the polypeptide and using the same cell withoutrecombinantly expressed polypeptide as a control. For example, SR-BI canbe expressed in BHK-21 or CHO cells using an expression vector, whereinthe same cell line without the expression vector can act as a control.Additional cell lines lacking SR-BI expression can be identified usingtechniques such as those employing SR-BI antibodies or those measuringSR-BI RNA.

A recombinant “nucleic acid” refers to an artificial combination of twoor more nucleotide sequence regions. The artificial combination is notfound in nature. Recombinant nucleic acid includes nucleic acid having afirst coding region and a regulatory element or a second coding regionnot naturally associated with the first coding region. The recombinantnucleotide sequence can be present in a cellular genome or can be partof an expression vector.

Preferably, expression is achieved in a host cell using an expressionvector. An expression vector contains recombinant nucleic acid encodinga polypeptide along with regulatory elements for proper transcriptionand processing. The regulatory elements that may be present includethose naturally associated with the nucleotide sequence encoding thepolypeptide and exogenous regulatory elements not naturally associatedwith the nucleotide sequence.

Generally, the regulatory elements that are present in an expressionvector include a transcriptional promoter, a ribosome binding site, aterminator, and an optionally present operator. Another preferredelement is a polyadenylation signal providing for processing ineukaryotic cells. Preferably, an expression vector also contains anorigin of replication for autonomous replication in a host cell, aselectable marker, a limited number of useful restriction enzyme sites,and a potential for high copy number. Examples of expression vectors arecloning vectors, modified cloning vectors, specifically designedplasmids and viruses.

An alternative means to produce recombinant nucleic acid is by alteringthe cellular genome. One type of alteration that can increase cellularexpression is the use of a strong promoter such as the immediate earlyhuman cytomegalovirus promoter. Alterations to the cellular genome canbe performed, for example, using techniques described by Ausubel,Current Protocols in Molecular Biology, John Wiley, 1987-1998.

Starting with a particular amino acid sequence and the known degeneracyof the genetic code, a large number of different encoding nucleic acidsequences can be obtained. The degeneracy of the genetic code arisesbecause almost all amino acids are encoded by different combinations ofnucleotide triplets or “codons”. Amino acids are encoded by codons asfollows:

-   A=Ala=Alanine: codons GCA, GCC, GCG, GCU-   C=Cys=Cysteine: codons UGC, UGU-   D=Asp=Aspartic acid: codons GAC, GAU-   E=Glu=Glutamic acid: codons GAA, GAG-   F=Phe=Phenylalanine: codons WTUC, UUU-   G=Gly=Glycine: codons GGA, GGC, GGG, GGU-   H=His=Histidine: codons CAC, CAU-   I=Ble=Isoleucine: codons AUA, AUC, AUU-   K=Lys=Lysine: codons AAA, AAG-   L=Leu=Leucine: codons UUA, WUG, CUA, CUC, CUG, CUU-   M=Met=Methionine: codon AUG-   N=Asn=Asparagine: codons AAC, AAU-   P=Pro=Proline: codons CCA, CCC, CCG, CCU-   Q=Gln=Glutamine: codons CAA, CAG-   R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU-   S=Ser=Serine: codons AGC, AGU, UCA, UCC, UCG, UCU-   T=Thr=Threonine: codons ACA, ACC, ACG, ACU-   V=Val=Valine: codons GUA, GUC, GUG, GUU-   W=Trp=Tryptophan: codon UGG-   Y=Tyr=Tyrosine: codons UAC, UAU

Nucleic acid having a desired sequence can be synthesized using chemicaland biochemical techniques. Examples of chemical techniques aredescribed in Ausubel, Current Protocols in Molecular Biology, JohnWiley, 1987-1998, and Sambrook et al., Molecular Cloning, A LaboratoryManual, 2^(nd) Edition, Cold Spring Harbor Laboratory Press, 1989.

Biochemical synthesis techniques involve the use of a nucleic acidtemplate and appropriate enzymes such as DNA and/or RNA polymerases.Examples of such techniques include in vitro amplification techniquessuch as PCR and transcription based amplification, and in vivo nucleicacid replication. Examples of suitable techniques are provided byAusubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998,Sambrook et al., Molecular Cloning, A Laboratory Manual, 2^(nd) Edition,Cold Spring Harbor Laboratory Press, 1989, and Kacian et al., U.S. Pat.No. 5,480,784.

Assay Formats

A variety of different assay formats may be employed to measureactivities related to HCV binding to SR-BI or a functional derivativethereof, activities related to HCV internalization, and activitiesrelated to functional surface expression of SR-BI or a functionalderivative thereof. Depending on the activity being assayed, assays canbe performed using whole cells, membrane preparations, and purifiedSR-BI.

Techniques for detecting binding to SR-BI include those employing a HCVE2 polypeptide containing a detectable label or region, and thoseinvolving the use of secondary antibodies to a region distinct from theSR-BI HCV E2 binding region. Assay formats that may be employed includeFACS analysis, a scintillation proximity assay (“SPA”), and sandwichtype assays where a detectable antibody is targeted to a region distinctfrom the SR-BI HCV E2 binding region. An example of techniques that canbe employed for a FACS analysis are provided in Example 1 infra.

Techniques for performing a SPA are well known in the art. SPA can beperformed using a bead or a plate coated with a scintillant fluid and aradiolabeled molecule. Proximity of the radiolabeled molecule to thescintillant fluid stimulates light emission. SPA can be used to measurebinding to SR-BI by, for example, joining SR-BI to a SPA bead or growingcells expressing SR-BI on a SPA plate (cytostar), radiolabeling a HCV E2polypeptide, and measuring the ability of a test compound to inhibitlight production from the radiolabeled polypeptide.

Antibodies binding to a region distinct from the SR-BI HCV E2 bindingsite can be employed to detect binding using capture assays formats. Forexample, cell membranes expressing SR-BI or purified SR-BI protein areattached to a solid support and a HCV E2 polypeptide is added to thesolid support in presence of the test compound, the solid support iswashed, and the presence of E2 on the support is determined using anantibody with a detectable label that binds to E2.

Activities related to HCV internalization can be assayed by, forexample, incubating cells with the HCV virus at 37° C. and measuring theintracytoplasmic HCV RNA by in situ hybridization (ISH). Examples of ISHtechniques are provided by Agnello et al., Proc. Natl. Acad. Sci. U.S.A.96:12766-12771,1999.

Activities related to functional surface expression of SR-BI or afunctional derivative thereof can be assayed by, for example, measuringsurface expression of SR-BI. Surface expression of SR-BI can be measuredusing compounds binding to SR-BI such as SR-BI antibodies or a labeledpolypeptide that binds to SR-BI.

Inhibition of SR-BI Expression

SR-BI nucleic acid activity can be inhibited using compounds affectingthe ability of such nucleic acid to be transcribed or translated.Inhibition of SR-BI nucleic acid activity can be used, for example, intarget validation studies, as a tool to study HCV infection, and toinhibit HCV infection or replication.

A preferred target for inhibiting SR-BI translation is mRNA. The abilityof mRNA encoding SR-BI to be translated into a protein can be affectedby compounds such as anti-sense nucleic acid and enzymatic nucleic acid.

Anti-sense nucleic acid can hybridize to a complementary region of atarget mRNA. Depending on the structure of the anti-sense nucleic acid,anti-sense activity can be brought about by different mechanisms such asblocking the initiation of translation, preventing processing of mRNA,hybrid arrest, and degradation of mRNA by RNAse H activity.

Enzymatic nucleic acid can recognize and cleave another nucleic acidmolecule. Preferred enzymatic nucleic acids are ribozymes. Ribozymestargeting particular nucleic acid motifs are well known in the art.

Modified and unmodified nucleic acids can be used as anti-sensemolecules and ribozymes. Different types of modifications can affectcertain anti-sense activities such as the ability to be cleaved by RNAseH, and can affect nucleic acid stability. Examples of referencesdescribing different anti-sense molecules and ribozymes, and the use ofsuch molecules, are provided in U.S. Pat. Nos. 5,849,902, 5,859,221, and5,852,188.

Administration

Compounds for treating HCV can be formulated and administered to apatient using the guidance provided herein along with techniques wellknown in the art. The preferred route of administration ensures that aneffective amount of compound reaches the target. Guidelines forpharmaceutical administration in general are provided in, for example,Remington's Pharmaceutical Sciences 18^(th) Edition, Ed. Gennaro, MackPublishing, 1990, and Modern Pharmaceutics 2^(nd) Edition, Eds. Bankerand Rhodes, Marcel Dekker, Inc., 1990, both of which are herebyincorporated by reference herein.

A “patient” refers to a mammal capable of being infected with HCV. Apatient may or may not be infected with HCV. Examples of patients arehumans and chimpanzees.

Depending upon the structure of a particular compound, the compound maybe prepared as an acidic or basic salt. Pharmaceutically acceptablesalts (in the form of water- or oil-soluble or dispersible products)include conventional non-toxic salts or the quaternary ammonium saltsthat are formed, e.g., from inorganic or organic acids or bases.Examples of such salts include acid addition salts such as acetate,adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate,butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, picrate, pival ate,propionate, succinate, tartrate, thiocyanate, tosyl ate, andundecanoate; and base salts such as ammonium salts, alkali metal saltssuch as sodium and potassium salts, alkaline earth metal salts such ascalcium and magnesium salts, salts with organic bases such asdicyclohexylamine salts, N-methyl-D-glucamine, and salts with aminoacids such as arginine and lysine.

Compounds can be administered using different routes such as byinjection. When administered by injection, the injectable solution orsuspension may be formulated using suitable non-toxic,parenterally-acceptable diluents or solvents, such as Ringer's solutionor isotonic sodium chloride solution, or suitable dispersing or wettingand suspending agents, such as sterile, bland, fixed oils, includingsynthetic mono- or diglycerides, and fatty acids, including oleic acid.Suitable dosing regimens are preferably determined taking into accountfactors well known in the art including type of subject being dosed;age, weight, sex and medical condition of the subject; the route ofadministration; the renal and hepatic function of the subject; thedesired effect; and the particular compound employed.

Optimal precision in achieving concentrations of drug within the rangethat yields efficacy without toxicity requires a regimen based on thekinetics of the drug's availability to target sites. This involves aconsideration of the distribution, equilibrium, and elimination of adrug. The daily dose for a patient is expected to be between 0.01 and1,000 mg per day.

EXAMPLES

Examples are provided below to further illustrate different features ofthe present invention. The examples also illustrate useful methodologyfor practicing the invention. These examples do not limit the claimedinvention.

Example 1 Materials and Methods

This example describes different materials and methods that wereemployed to study HCV E2 binding to SR-BI.

Cells

Molt-4 (human T-cell leukaemia) cells were obtained from the MRC ADPRepository. HepG2 (human hepatoma), HEK-293 (human embryonic kidney)BHK-21 (baby hamster kidney) and CHO (chinese hamster ovary) cell lineswere obtained from the ATCC Repository. Cells were grown under standardconditions in presence of 10% fetal calf serum.

Cloning and Expression of E2 Glycoproteins

HCV E2 protein representative of genotype 1a (strain H) and genotype 1b(strain BK) were cloned into VIJnsTPA as described by Meola et al., J.Virol. 74:5933-5938, 2000. The cloned E2 fragment contains the codingsequence for E2 from amino acid 384 to amino acid 661 of the HCVpolyprotein and a tag of 6 His at the C-terminal. 293 cells weretransfected by calcium phosphate method. Recombinant proteins producedby transfected HEK 293 cells were collected from culture supernatantharvested 48 hours after transfection, concentrated 40 times usingfilter devices (Millipore Centricon Plus-80) and supplemented withprotease inhibitor cocktail tablets (Boehringer Mannheim) and 10%glycerol. The amount of E2 in the extracts was quantified by using a GNA(lectin from Galanthus nivalis) capture assay as described by Flint etal., J. Virol. 73:6782-6790, 1999.

FACS Analysis of Anti-CD81 and E2 Glycoproteins Binding to Cell Lines

Cells were washed twice in phosphate-buffered saline 1% FCS, 0.05%sodium azide (washing buffer). Then, 2×10⁵ cells were allowed to bind atroom temperature for 1 hour with E2 concentrated supernatants or, ascontrol, with the concentrated supernatant of HEK 293 cells transfectedwith VIJnsTPA plasmid (mock). After one wash in washing buffer theanti-His mouse monoclonal antibody (mAb, Quiagen) was added at theconcentration of 2 μg/ml for 1 hour at room temperature. Binding toanti-CD81 was also performed at room temperature for 1 hour by using themouse monoclonal antibody (1.3.3.22 Santa Cruz Biotechnology) diluted1:200.

Cell bound mAbs were visualized with anti-mouse IgG1-phycoerythrin (PE)conjugate (Serotec). Flow cytometry data acquisition was performed usinga Becton-Dickinson FACS Vantage flow cytometer. Dead cells were detectedas Sytox Green dye (Molecular Probes) stained positive and were excludedfrom analyses.

Selection of a HepG2 Sub-Population with Increased E2-Binding Capacity

HepG2 cells were incubated with a saturating concentration of 1a-derivedE2 recombinant protein for 2 hours at room temperature. The E2 boundprotein was revealed as described above and analyzed in a FACS Vantage(Becton Dickinson) flow cytometer. The cells that showed die highestfluorescence intensity were sorted and expanded. The procedure wasrepeated four times.

Coating of Anti-His Antibody onto Dynabeads

Dynabeads M-450 rat anti-mouse IgG1, (Dynal, Oxoid) were washed oncewith PBS 0.1% BSA before the addition of 15 μg of the anti-His Mab in 4ml volume of PBS 0.1% BSA. After 1 hour of coating, the beads werewashed twice with PBS 0.1% BSA and once with 0.2 M triethanolamine pH 9.To cross-link the bound antibody dimethylpimlimidate dihydrochlorite(DMP) was added at a 20 mM concentration to the Dynabeads in 10 mlvolume of 0.2 M triethanolamine pH 9. The reaction was stopped by adding0.2 M Tris HCl at pH 8. Beads were washed first with PBS 0.5% Triton andthen with PBS 0.1% BSA. Once prepared the Dynabeads were stored at 4° C.in presence of 0.1% sodium azide.

Cell Surface Biotinylation

HepG2s4 cells (1×10⁸) were harvested by trypsinization and washed oncewith cold PBS in 250 ml volume. Cells were incubated in the dark for 15minutes with a freshly prepared solution of 2 mM sodium periodate at theconcentration of 1×10⁷ cell/ml. After this mild oxidation, cells werewashed twice and incubated with 5 mM biotin-LC hydrazide for 10 minutesat room temperature at the concentration of 5×10⁷ cell/ml. Biotin-LChydrazide was freshly prepared by solubilization in DMSO at a 50 mMconcentration.

After biotinylation two washes in 250 ml volume of cold PBS wereperformed, and cells were incubated with the concentrated supernatantcontaining E2 recombinant protein or as a control with the supernatantobtained from the mock transfection. Incubation with E2 lasted 1 hour atroom temperature. Staining of bound E2 was performed as described aboveand analyzed by FACS.

E2-Receptor Cross-Linking and Immunoprecipitation of Complexes

DTSSP (dithiobis-sulfosuccinimidylpropionate, Pierce,) was used as across linker. DTSSP is a water soluble cross-linker homobifunctionalN-hydroxysuccinimide ester and is thiol cleavable.

HepG2s4 cells (5×10⁷ cell/ml) were washed once after binding to E2 andincubated with the DTSSP at 2 mM concentration in PBS for 20 minutes atroom temperature. The reaction was stopped by incubation with Tris HCl50 in M at pH 7.5. Cells were lysed in PBS 1% Triton in the presence ofprotease inhibitor cocktail (Boehringer Mannheim) for 20 minutes at 37°C. E2-receptor complexes were immunoprecipitated with the anti-HisDynabeads by incubation overnight at 4° C. After 5 washes in PBS 1%Triton elution was performed either boiling directly the beads in sodiumdodecyl sulfate (SDS) sample buffer or incubating beads 30 minutes at37° C. in 50 mM dithiothreitol (DTT), 50 mM NaCl, 50 mM Tris HCl pH 8.

Samples obtained from boiling the beads were loaded on SDS-Page andanalyzed by Western blot for detection of biotinylated proteins usingstreptavidin horseradish peroxidase conjugated (HRP; Pierce) diluted1:25.000 in Tris Buffer Saline 0.05% Tween 20 (TBST), 2% bovine serumalbumin (BSA). For detecting recombinant E2 protein of genotype 1a therat monoclonal antibody 6-1/a (Flint et al., J. Virol. 73:6782-6790,1990) was used diluted 1:50 in TBST 5% low fat milk, followed byincubation with anti-rat HRP (Dako) conjugated 1:2000. Thechemiluminescent Super Signal West Pico (Pierce) substrate was used andimmunoreactive proteins were detected by exposure on X ray film (KodakBiomax ML).

Con-A Sepharose Purification and Enzymatic Deglycosylation of the E2Receptor

Eluates obtained from the immunoprecipitation with anti-His Dynabeadswere diluted 1:2 with 1 M NaCl, 0.2% Triton and incubated with Con-Abeads for two hours at room temperature. After three washes inincubation buffer, elution was performed under denaturing conditions in1% SDS, 1% β-mercaptoethanol and 100 mM phosphate pH 7.5 at 95° C. for10 minutes. The eluates were diluted 1:10 in the PNGase F incubationbuffer containing 0.1% SDS, 0.5% NP40, 10 nM EDTA, and 100 mM Naphosphate pH 7.5. Eluates were divided into aliquots one of which wastreated with the enzyme PNGase F (Bio-rad) for 3 hours at 37° C.

Samples were loaded on 7.5% SDS pre-cast gel (Bio-rad) and silverstained. Immunoreactivity of the purified samples was probed in Westernblot by using rabbit anti-SR-BI purified polyclonal antibodies (NovusBiologicals NB 400-104) diluted 1:1500 in TBST 2% BSA followed byanti-rabbit HRP (Pierce) diluted 1: 20.000. The chemiluminescentsubstrate Super Signal West Pico (Pierce) was used for detection.

Cloning of SR-BI Coding Sequence and Transfection in BHK Cell Line

RNA was extracted from HepG2s4 cells with TRIzol reagent (Gibco BRL)following manufacturer's instructions. First-strand cDNA was producedmixing total RNA (2 μg) with 10 pmol of the antisense primer SR-BI5′-CCAGTCTAGACAGTTTTGCTTCCTGCAGCACAGAGCCC-3′ (SEQ. ID. NO. 3). Therestriction site for Xba I is in italics. The reaction was performed byusing Superscript II reverse transcriptase (Gibco BRL) as described byMeola et al., J. Virol 74:5933-5938, 2000.

An aliquot of the cDNA was amplified by PCR using Platinum Pfx DNApolymerase (Gibco BRL) following manufacturer's instructions. Theprimers used were the antisense SR-BI primer and the sense SR-BI5′-AGGCAAGCTTGCCGCCATGGGCTGCTCCGCCAAAGCGCGCTGGG-3′, (SEQ. ID. NO. 4)where the restriction site for Hind III is in italics. PCR was performedin a Perkin-Elmer 2400 thermocycler, denaturing samples for 4 minutes at94° C. and then running 35 cycles of incubation at 94° C. for 30seconds, at 50° C. for 30 seconds, and 68° for 2 minutes.

The PCR product was digested with the restriction enzymes Hind III andXba I for directional cloning in the vector pcDNA3. Clones weresequenced with the Big Dye Terminator Cycle Sequencing Kit usingAmpliTaq (Applied Biosystems) and an Applied Biosystem Model 373ASequencer. Sequences were analyzed by the Vector NTI program.

The BHK-21 cell line was used as recipient for transient transfection.Transfection was performed by mixing plasmid DNA with the FuGENE 6transfection reagent (Roche). Cells were harvested 24 hours aftertransfection and analyzed by FACS for E2 binding.

Example 2 Identification of SR-BI as an E2 Receptor

SR-BI was identified as an E2 receptor by examining the ability of E2 tobind to HepG2 cells, enriching for HepG2 cells having increased abilityto bind E2, and determining that SR-BI binds E2. HepG2 cells were usedto search for the E2 receptor because they were found to lack CDS81 andretain the ability to bind HCV E2.

Characterization of CD81-Independent Binding to E2 Glycoproteins

The ability of HepG2 cells to bind E2 independent of CD81 was determinedusing a FACS analysis. The absence of the CD81 molecule from HepG2 cellswas determined using an anti-CD81 mouse monoclonal antibody, and asecondary antibody anti-mouse IgG1-phycoerythrin conjugate. Binding ofrecombinant E2 proteins derived from genotype 1a and 1b to cells wasdetected by antibodies reactive against a 6-His tag present in therecombinant proteins followed by incubation with an anti-mouseIgG1-phycoerythrin conjugate.

FACS analysis for CD81 expression was performed using HepG2 cells andMolt-4 cells as described in Example 1. HepG2 cells were negative forCD81 expression. In contrast, Molt-4 cells showed high levels of CD81 onthe cell surface.

FACS analysis for E2 binding was performed using HepG2 cells and Molt4cells as described in Example 1. Both cell lines tested showed bindingto recombinant E2 derived from genotype 1a. However, the E2 recombinantglycoprotein derived from 1b genotype showed a much reduced binding toMolt-4 cells, while the binding to HepG2 cells was comparable to thatobtained with 1a derived E2 recombinant protein.

Cross-Linking of Recombinant E2 Protein to the Cellular Receptor

Enrichment of HepG2 cells expressing high levels of the E2 receptor wasachieved with four subsequent rounds of sorting. A subpopulation ofHepG2 cells “HepG2s4” showed upon binding to E2, a mean fluorescenceintensity value 3.5 times higher than the original cell population. Theobserved phenotype was stable after several weeks of cell culture.

Surface labeling of HepG2s4 was performed using a biotin-LC-hydrazidereagent. (Kahne et al., J. ImmunoL Methods. 168:209-218, 1994.) Thehydrazides are reactive with the aldehyde groups obtained by mildoxidation with sodium periodate of hydroxyl groups of the glycoproteincarbohydrate moieties. This biotinylation method is compatible with thesubsequent step of cross-linking involving a primary amine as a targetfor a NHS(N-hydroxisuccinimide) ester cross-linker.

Biotinylated HepG2s4 cells were incubated with the E2 glycoprotein 1a.The reactivity of E2 to the putative receptor was unaffected by thebiotinylation procedure as detected by flow cytometric analysis afterstaining of bound E2.

Cross-linking was performed after the E2 binding by addition of theDTSSP cross-linker, that is cleavable by thiol. Cells were finally lysedin PBS 1% Triton and the E2-receptor complexes were immunoprecipitatedunder non-reducing conditions by Dynabeads conjugated with an antibodyreactive against the His tag of recombinant E2. The experiment was runin parallel with a control sample where the biotinylated HepG2s4 cellswere incubated with the concentrated supernatant from the mocktransfected 293 cells before the cross-linking.

Immunoprecipitated samples were eluted directly in sample buffer bothunder reducing and non-reducing conditions and loaded on SDS-page.Immunoblot was performed by using an anti-82 rat monoclonal antibody,6/1a, specific for genotype 1a protein (Flint et al., J. Virol.73:6782-6790, 1990), followed by an anti-rat secondary antibodyconjugated with peroxidase for enhanced chemiluminescence detection.

E2 protein was detected as a diffuse band of the expected molecularweight for the monomer species under reducing condition. Undernon-reducing conditions most of E2 protein was detected at highermolecular weight probably representing the E2 receptor-complexes andadditional aggregated forms of E2 (FIG. 1A).

For detection of biotinylated species Western blot were developed withstreptavidin peroxidase conjugate. By thiol cleavage of the E2/receptorcomplexes, it was possible to detect only under reducing condition apredominant biotinylated band with an apparent molecular weight of 82kDa, probably corresponding to the biotinylated receptor (FIG. 1B).

Purification and Enzymatic Deglycosylation of the Putative Receptor

The cross-linking experiment indicated that the receptor involved in E2binding was an 82 kd glycoprotein, since only glycoproteins could havebeen biotinylated by the biotin hydrazide reagent. The protein involvedin E2 binding was identified by performing cross-linking in the absenceof the surface labeling step with biotin due to cellular toxicityassociated with sodium periodate. Treatment with sodium periodate wasquite toxic for the cells increasing significantly the number of cellslost before binding.

Cells were harvested (6×10⁸ cells) to perform binding to E2 followed bycross-linking. The E2/receptor complexes were purified from the lysatewith anti-His Dynabeads as described in Example 1, and eluting thereceptor molecule from the complex by incubation in 50 mM DTT at 37° C.for 30 minutes. The analysis of the eluate on SDS page by silverstaining indicated that several molecular species were eluted togetherwith the putative receptor.

A second purification step was performed using Concanavalin A lectin(Con-A). Con-A is a commonly used lectin for purification ofglycoproteins that binds Asn-linked glycans. Samples eluted from theanti-His Dynabeads were incubated with Con-A sepharose for a secondimmunoprecipitation.

After several washes in the incubation buffer, elution from the Con-Abeads was performed under denaturing condition compatible with theenzymatic activity of PNGase F. PNGas F is an enzyme that releases allAsn-linked oligosaccharides from the glycoproteins.

The eluted sample was enzymatically deglycosylated by using the PNGase Fenzyme. Analysis of samples on silver stained gel visualized theglycosylated receptor band with an apparent molecular weight of 82 kDaand the deglycosylated form migrating at 54 kDa compatible with thepresence of 10 potential Asn glycosylation sites (FIG. 2).

Identification of the Putative Receptor

Based on preliminary data, SR-BI was suspected of being an HCV receptor.Preliminary data on inhibition of the E2 binding to HepG2 cells withβ-cyclodextrins suggested that cholesterol may play a role in theobserved binding. β-Cyclodextrins can selectively remove cholesterolfrom cell membranes. (Yancey et al., J. Biol. Chem. 271:16026-16034,1996.) Additionally, the migration pattern on SDS page of theglycosylated and deglycosylated receptor were very similar to that forSR-BI.

SR-BI was confirmed to be the receptor binding HCV E2 using anti-SR-BIantibodies. The reactivity of the purified proteins in Western blot withantibodies against SR-BI is shown in FIG. 3.

Example 3 Transfection of the SR-BI Coding Sequence in BHK-21 Cell Line

The coding sequence for the human SR-BI was amplified from RNA ofHepG2s4 cells and cloned in a vector suitable for transfection.Transfection was performed in BHK-21 recipient cells since they werenegative for E2 binding. FACS analysis of cells 24 hours aftertransfection indicated that SR-BI transfected cells acquired binding forE2 (FIG. 4).

Example 4 Transfection of Human and Mouse SR-BI Coding Sequence in CHOCell Line

The human SR-BI coding sequence in Example 3 was amplified with thesense primer (SEQ. ID. NO. 4) and the antisense primer (SEQ. ID. NO. 3).The stop codon in this construct was provided by the cloning vector 36nucleotides after the SR-BI coding sequence, and therefore 12 aminoacids were added to the carboxyl terminal of the SR-BI natural sequence.To obtain the human SR-BI protein of its natural size ending with theleu 509 (SEQ. ID. NO. 1), the coding sequence for human SR-BI was PCRamplified by using the sense primer (SEQ. ID. NO. 4) and a novelantisense primer (SEQ. ID. NO. 5). The new primer contains a stop codonin the primer sequence.

The mouse SR-BI sequence was amplified from IMAGE clone BC004656 byusing the sense primer (SEQ.ID. NO. 6), and the antisense primer (SEQ.ID. NO. 7). The human and the mouse sequences were cloned in pcDNA3vector and clones obtained were sequenced.

The hamster CHO cell line negative for binding to HCV E2 protein wastransfected with the plasmids using lipofectamine 2000 reagent(Invitrogen). The combination of CHO cells and lipofectamine reagentgave improved transfection efficiency. Transfected cells were harvested24 hours after transfection and analyzed by FACS, for receptorexpression and E2 binding capability.

Results demonstrate that the human and the mouse receptors, wereexpressed at comparable levels (FIGS. 5A-C), but only cells transfectedwith the human SR-BI acquired the ability to bind HCV E2 (FIGS. 5D-F).Moreover, the mouse SR-BI, showing 80% of homology at amino acid levelto the human receptor, doesn't bind to HCV E2, mirroring the speciesspecificity of HCV infection (FIGS. 5D-F).

Example 5 A Monoclonal Antibody Against the Hypervariable Region 1(HVR1) of HCV E2 Glycoprotein Inhibits the E2 Binding to SR-BI.

A biological relevant question concerns the ability of antibodiesagainst the hypervariable region 1 to neutralize HCV virus. A monoclonalantibody (9/27; Flint, et al., 2000, J. Virol., 74, 702-709), obtainedupon immunization with E2 and reactive against the HVR1 of E2 derivedfrom H isolate was used to inhibit E2 binding to SR-B1. The antibodyshowed a dose dependent inhibitory activity for the binding of the E2protein from genotype 1a to HepG2 cells and to CHO cells stablytransfected with SR-BI with an apparent IC₅₀ of about 500 nM (FIG. 6).The antibody was not effective on the binding of the E2 protein derivedfrom genotype 1b, BK strain, consistent with its lack of reactivity withthis variant (FIG. 6).

Other embodiments are within the following claims. While severalembodiments have been shown and described, various modifications may bemade without departing from the spirit and scope of the presentinvention.

1. A method of screening for a compound that inhibits the ability of ahepatitis C virus E2 polypeptide to bind to a cell comprising the stepsof: a) contacting a human scavenger receptor class B type I (SR-BI) orfunctional derivative thereof with a polypeptide that binds to the SR-BIHCV E2 binding site and with a test compound, and b) measuring theability of said test compound to inhibit binding of said polypeptide tosaid SR-BI.
 2. The method of claim 1, wherein said SR-BI is present as asoluble protein.
 3. The method of claim 1, wherein said SR-BI is presentin a membrane preparation.
 4. A method of screening for a compound thatinhibits SR-BI activity comprising the steps of: a) contacting a cellcapable of expressing a human SR-BI or functional derivative thereofwith a polypeptide that binds to the HCV SR-BI E2 binding site and witha test compound, and b) measuring the ability of said test compound toinhibit one or more of the following: (i) binding of said polypeptide tosaid SR-BI or functional derivative thereof, (ii) HCV internalization,and (iii) functional surface expression of said SR-BI or functionalderivative thereof.
 5. The method of claim 4, wherein said cell ispre-incubated with said test compound prior to adding said polypeptide.6. The method of claim 4, wherein said SR-BI or functional derivativethereof has an amino acid sequence substantially similar to SEQ IDNO:
 1. 7. The method of claim 6, wherein said step (b) measures theability of said test compound to inhibit binding of said polypeptide tosaid cell.
 8. The method of claim 7, wherein said cell comprisesrecombinant nucleic acid capable of expressing said SR-BI or functionalderivative thereof.
 9. The method of claim 8, wherein said cell is amammalian cell.
 10. The method of claim 9, wherein said mammalian celldoes not endogenously express said SR-BI or functional derivativethereof.
 11. The method of claim 10, wherein said SR-BI is the humanSR-BI of SEQ ID NO:
 1. 12. The method of claim 11, wherein saidpolypeptide comprises a naturally occurring E2 region.
 13. The method ofclaim 12, wherein said polypeptide comprises a E2 region from either HCV1a or HCV 1b.
 14. A method of inhibiting entry of a hepatitis C virusinto a cell comprising the step of contacting said cell with an SR-BI E2binding antagonist.
 15. The method of claim 14, further comprising thestep of using said antagonist as the test compound in the method ofclaim 1 prior to inhibiting entry of said hepatitis C virus.
 16. Amethod of treating an HCV infected patient comprising the step ofdecreasing SR-BI activity or functional surface expression.
 17. Themethod of claim 16, wherein method inhibits the ability of SR-BI to bindHCV.
 18. The method of claim 16, wherein said step of decreasing SR-BIactivity or functional surface expression is performed using a compoundidentified as inhibiting binding to SR-BI or a functional derivativethereof or inhibiting SR-BI activity using the method of claim 1.